Vascular biology
A 4-week high-AGE diet does not impair glucose metabolism and vascular function in obese individuals
Abstract
BACKGROUND. Accumulation of advanced glycation endproducts (AGEs) may contribute to the pathophysiology of type 2 diabetes and its vascular complications. AGEs are widely present in food, but whether restricting AGE intake improves risk factors for type 2 diabetes and vascular dysfunction is controversial. RESEARCH DESIGN AND METHODS. Abdominally obese but otherwise healthy individuals were randomly assigned to a specifically designed 4-week diet low or high in AGEs in a double blind parallel-design. Insulin sensitivity, secretion, and clearance were assessed by a combined hyperinsulinemic-euglycemic and hyperglycemic clamp. Micro- and macrovascular function, inflammation, and lipid profile were assessed by state-of-art in vivo measurements and biomarkers. Specific urinary and plasma AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were assessed by mass spectrometry. RESULTS. In 73 individuals (22 males, mean ± SD age and BMI 52 y ± 14, 30.6 kg/m2 ± 4.0), intake of CML, CEL, and MG-H1 differed 2.7, 5.3, and 3.7-fold between the low and high AGE diets, which led to corresponding changes of these AGEs in urine and plasma. Despite this, there was no difference in insulin sensitivity, secretion, or clearance, micro- and macrovascular function, overall inflammation, or lipid profile between the low and high dietary AGE groups (all p for treatment effects > 0.05). CONCLUSIONS. This comprehensive RCT demonstrates very limited biological consequences of a 4-week diet low or high in AGEs in abdominally obese individuals. TRIAL REGISTRATION. clinicaltrials.gov: NCT03866343, trialregister.nl: NTR7594. FUNDING. Diabetesfonds and ZonMw.
Authors
Armand M A Linkens, Alfons J. Houben, Petra M Niessen, Nicole Wijckmans, Erica de Goei, Mathias D.G. Van den Eynde, Jean L. J. M. Scheijen, Marjo Waarenburg, Andrea Mari, Tos T.J.M. Berendschot, Lukas Streese, Henner Hanssen, Martien C.J.M. van Dongen, Christel van Gool, Coen D.A. Stehouwer, Simone JPM Eussen, Casper Schalkwijk.
Abstract
Capillary malformation-arteriovenous malformation (CM-AVM) is a blood vascular anomaly caused by inherited loss of function mutations in RASA1 or EPHB4 genes that encode p120 Ras GTPase-activating protein (p120 RasGAP/RASA1) and Ephrin receptor B4 (EPHB4) respectively. However, whether RASA1 and EPHB4 function in the same molecular signaling pathway to regulate the blood vasculature is uncertain. Here, we show that induced endothelial cell (EC)-specific disruption of Ephb4 in mice results in accumulation of collagen IV in the EC endoplasmic reticulum leading to EC apoptotic death and defective developmental, neonatal and pathological angiogenesis, as reported previously in induced EC-specific RASA1-deficient mice. Moreover, defects in angiogenic responses in EPHB4-deficient mice can be rescued by drugs that inhibit signaling through the Ras pathway and drugs that promote collagen IV export from the ER. However, EPHB4 mutant mice that express a form of EPHB4 that is unable to physically engage RASA1 but retains protein tyrosine kinase activity show normal angiogenic responses. These findings provide strong evidence that RASA1 and EPHB4 function in the same signaling pathway to protect against the development of CM-AVM independent of physical interaction and have important implications with regards possible means of treatment of this disease.
Authors
Di Chen, Elizabeth D. Hughes, Thomas L. Saunders, Jiangping Wu, Magda N. Hernández Vásquez, Taija Makinen, Philip D. King
Abstract
Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian hemoglobins (Hb), β-globin Cys93 (βCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during oxygen delivery. βCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting oxygen normally. Here we show that βCys93Ala mutant mice have reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion, and that hypoxic vasodilation by RBCs is impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, βCys93Ala mutant mice show marked impairment of hypoxic peripheral vasodilation and develop signs of pulmonary hypertension with age. Mutant mice also die prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low oxygen. Altogether, we identify a major role for RBC-SNO in clinically-relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.
Authors
Rongli Zhang, Alfred Hausladen, Zhaoxia Qian, Xudong Liao, Richard T. Premont, Jonathan S. Stamler
Abstract
The anatomical routes for the clearance of cerebrospinal fluid (CSF) remain incompletely understood. However, recent evidence has given strong support for routes leading to lymphatic vessels. A current debate centers upon the routes through which CSF can access lymphatics, with evidence emerging for either direct routes to meningeal lymphatics or along cranial nerves to reach lymphatics outside the skull. Here, a method was established to infuse contrast agent into the ventricles using indwelling cannulae during imaging of mice at 2 and 12 months of age by magnetic resonance imaging. As expected, a significant decline in overall CSF turnover was found with aging. Quantifications demonstrated that the bulk of the contrast agent flowed from the ventricles to the subarachnoid space in the basal cisterns. Comparatively little contrast agent signal was found at the dorsal aspect of the skull. The imaging dynamics from the two cohorts revealed that the contrast agent cleared from the cranium through the cribriform plate to the nasopharyngeal lymphatics. On decalcified sections, we confirmed that fluorescentlylabeled ovalbumin drains through the cribriform plate and can be found within lymphatics surrounding the nasopharynx. In conclusion, routes leading to nasopharyngeal lymphatics appear to be a major efflux pathway for cranial CSF.
Authors
Yann Decker, Jonas Krämer, Li Xin, Andreas Müller, Anja Scheller, Klaus Fassbender, Steven T. Proulx
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options. Despite endothelial cells (ECs) comprising 30% of the lung cellular composition, the role of EC dysfunction in pulmonary fibrosis (PF) remains unclear. We hypothesize that sterol regulatory element-binding protein 2 (SREBP2) plays a critical role in the pathogenesis of PF via EC phenotypic modifications. Transcriptome data demonstrate that SREBP2 overexpression in ECs led to the induction of the TGF, Wnt, and cytoskeleton remodeling gene ontology pathways and the increased expression of mesenchymal genes, such as snail family transcriptional repressor 1 (snai1), α-smooth muscle actin, vimentin, and neural cadherin. Furthermore, SREBP2 directly bound to the promoter regions and transactivated these mesenchymal genes. This transcriptomic change was associated with an epigenetic and phenotypic switch in ECs, leading to increased proliferation, stress fiber formation, and ECM deposition. Mice with endothelial-specific transgenic overexpression of SREBP2 (EC-SREBP2[N]-Tg mice) that were administered bleomycin to induce PF demonstrated exacerbated vascular remodeling and increased mesenchymal transition in the lung. SREBP2 was also found to be markedly increased in lung specimens from patients with IPF. These results suggest that SREBP2, induced by lung injury, can exacerbate PF in rodent models and in human patients with IPF.
Authors
Marcy Martin, Jiao Zhang, Yifei Miao, Ming He, Jian Kang, Hsi-Yuan Huang, Chih-Hung Chou, Tse-Shun Huang, Hsiao-Chin Hong, Shu-Han Su, Simon S. Wong, Rebecca L. Harper, Lingli Wang, Rakesh Bhattacharjee, Hsien-Da Huang, Zhen Bouman Chen, Atul Malhotra, Marlene Rabinovitch, James S. Hagood, John Y-J. Shyy
Abstract
Inhibitors of the renin-angiotensin system (RAS) are widely used to treat hypertension. Using mice harboring fluorescent cell lineage tracers, single-cell RNA-seq, and long-term inhibition of RAS in both mice and humans, we found that deletion of renin or inhibition of the RAS leads to concentric thickening of the intrarenal arteries and arterioles. This severe disease is caused by the multiclonal expansion and transformation of renin cells from a classical endocrine phenotype to a matrix-secretory phenotype: the cells surround the vessel walls and induce the accumulation of adjacent smooth muscle cells and extracellular matrix, resulting in blood flow obstruction, focal ischemia, and fibrosis. Ablation of the renin cells via conditional deletion of β1integrin prevents arteriolar hypertrophy, indicating that renin cells are responsible for vascular disease. Given these findings, prospective morphological studies in humans are necessary to determine the extent of renal-vascular damage caused by the widespread use of inhibitors of RAS.
Authors
Hirofumi Watanabe, Alexandre G. Martini, Evan A. Brown, Xiuyin Liang, Silvia Medrano, Shin Goto, Ichiei Narita, Lois J. Arend, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
Abstract
Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR, Cx40, and Nox4 levels were decreased in CECs from diabetic and HuR-KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40-KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.
Authors
Rui Si, Jody Tori O. Cabrera, Atsumi Tsuji-Hosokawa, Rui Guo, Makiko Watanabe, Lei Gao, Yun Sok Lee, Jae-Su Moon, Brian T. Scott, Jian Wang, Anthony W. Ashton, Jaladanki N. Rao, Jian-Ying Wang, Jason X.-J. Yuan, Ayako Makino
Abstract
Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP–treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype–associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.
Authors
J. William Tierney, Brian C. Evans, Joyce Cheung-Flynn, Bo Wang, Juan M. Colazo, Monica E. Polcz, Rebecca S. Cook, Colleen M. Brophy, Craig L. Duvall
Abstract
We recently described a previously unknown trans-tentorial venous system (TTVS) connecting venous drainage throughout the brain in humans. Prior to this finding, it was believed that the embryologic tentorial plexus regresses, resulting in a largely avascular tentorium. Our finding contradicted this understanding and necessitated further investigation into the development of the newly described TTVS. Herein we sought to investigate mice as a model to study the development of this system. First, using vascular casting and ex vivo micro-computed tomography (micro-CT), we demonstrate that this TTVS is conserved in adult mice. Next, using high-resolution magnetic resonance imaging (MRI), we found the primitive tentorial venous plexus in murine embryo at day 14.5. We also found that, at this embryologic stage, the tentorial plexus drains the choroid plexus. Finally, using vascular casting and micro-CT, we found that the TTVS is the dominant venous drainage in the early postnatal period (P8). Herein, we demonstrate that the TTVS is conserved between mice and humans and present a longitudinal study of its development. In addition, our findings establish mice as a translational model for further study of this newly described system and its relationship to intracranial physiology.
Authors
Pashayar P. Lookian, Vikram Chandrashekhar, Anthony Cappadona, Jean-Paul Bryant, Vibhu Chandrashekhar, Jessa M. Tunacao, Danielle R. Donahue, Jeeva P. Munasinghe, James G. Smirniotopoulos, John D. Heiss, Zhengping Zhuang, Jared S. Rosenblum
Abstract
Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2-angiopoietin axis. Primary human endothelial cells treated with plasma from patients with severe COVID-19 upregulated expression of thromboinflammatory genes, inhibited expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from COVID-19 patients demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity and highest levels were associated with worse survival. These data highlight the disruption of Tie2-angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.
Authors
Alec A. Schmaier, Gabriel M. Pajares Hurtado, Zachary J. Manickas-Hill, Kelsey D. Sack, Siyu M. Chen, Victoria Bhambhani, Juweria Quadir, Anjali K. Nath, Ai-ris Y. Collier, Debby Ngo, Dan H. Barouch, Nathan I. Shapiro, Robert E. Gerszten, Xu Yu, Kevin G. Peters, Robert Flaumenhaft, Samir M. Parikh
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